Hundreds of genetic variants underlying risk for type 2 diabetes (T2D) and variation in T2D-related quantitative traits have been identified, but there has been little focus on how these variants alter basic physiology to contribute to the pathophysiology of disease. The over-arching goal of this grant is to establish a research program focused on assessing the physiologic effect of genetic variation in vivo in humans and determining how variation alters physiologic processes to contribute to disease pathogenesis. In this application we specifically focus on how genetic variation in glucokinase (GCK), glucokinase regulator (GCKR), and patatin-like phospholipase domain-containing protein 3 (PNPLA3) might determine the level of liver fat and subsequent hepatic insulin resistance. We propose the hepatic substrate balance hypothesis in which genetic variation contributes to an imbalance in substrate flow into and out of the liver. GCK is responsible for hepatic glucose uptake and GCKR regulates the enzymatic activity of GCK. Thus, one could imagine genetic variation may alter the individual activities of GCK and GCKR, or their interaction, which then alters hepatic glucose uptake, and the conversion of glucose to fat. PNPLA3 hydrolyzes fat to triglyceride and genetic variation could alter the efficiency of PNPLA3 to break down fat in the liver. The amount of fat within the liver is a balance between these two processes and imbalance results in the accumulation of liver fat, which in turn results in hepatic insulin resistance. We also propose the hypothesis that dietary fructose, which itself contributes to liver fat, may also exacerbate the substrate imbalance due to genetic variation through its regulation of GCKR. We propose three aims in this study. In Aim 1 we will recruit 265 new participants and perform detailed phenotyping as previously performed, but include an assessment of liver fat by MRI. These studies will be used to validate preliminary observations of associations between genetic variants and liver enzyme levels. In Aim 2 we proposed to analyze lactate kinetics from the FSIGT to obtain measures of GCK activity and hepatic glycolytic flux. We will test these phenotypes for association with genetic variants to validate our preliminary observation of association between genetic variants and glucose effectiveness and liver enzyme levels. We will also examine the interaction with dietary fructose, since it is known that fructose-1-phosphate regulates GCKR activity. In Aim 3, we propose to test genetic variation from not just these three loci, but also other loci showing evidence for association with hepatic steatosis or liver enzyme levels. In particular, we will focus on gene-gene interaction and gene-dietary fructose interactions to build a comprehensive model of our substrate balance hypothesis.